The use of populations of cells with antitumor cell activity is an attractive alternative to standard chemotherapy for patients with cancer. Previously, we have reported a novel protocol for the generation of highly efficient cytotoxic effector cells by culturing PBLs in the presence of IFN-gamma on day 1, and IL-2, mAB-CD3 and IL-1 on the following day. We have termed these cultures cytokine-induced killer (CIK) cells, inasmuch as the phenotype of the cells with the greatest cytotoxic activity expresses both the T cell marker CD3 and the NK cell marker CD56. CD3+CD56+ cells are rare (1 about 5%) in uncultured PBLs, yet have been demonstrated to have MHC-unrestricted cytotoxicity. Recently, we have reported that under our culture conditions, the CD3+CD56+ cells expand nearly 1000-fold, of which the majority are derived from CD3_CD56- T cells, and not from CD3- CD56+ NK cells. The CIK cells have more potent antitumor activity as compared to lymphokine-activated killer cells in mice with severe combined immunodeficiency (SCID) and are more easily accessible than tumor infiltrating lymphocytes. In this proposal we will optimize the expansion and cytotoxicity of CD3+CD56+ cells (CIK effectors). We will evaluate whether growth factor and cytoxan-mobilized peripheral blood contains a larger number of precursor cells which are capable of expansion into CD3+CD56+ killer cells than steady state peripheral blood. We will investigate and characterize the functional properties of expanded CD3+CD56+ cells and their target cell specificity at the clonal level. Finally, we will utilize SCID mice as model to study in vivo antitumor activity of CIK cells under several conditions. We believe that by further study of the major effector cells in CIK culture, namely CD3+CD56+ cells, we will not only gain fundamental biological insights but also develop new treatment approaches for patients with cancer.